MXPA06008918A - Methods of treating skin disorders. - Google Patents

Abstract

Methods of treating skin disorders are provided.

Description

METHODS OF TREATMENT OF SKIN DISORDERS FIELD OF THE INVENTION The present invention concerns methods of treating a variety of conditions, including conditions mediated by T cells. BACKGROUND OF THE INVENTION Psoriasis affects 4.5 million adults in the United States, AMEVIVE® (Alefacept) is an agent approved for use in treating psoriasis. SUMMARY OF THE INVENTION The invention provides methods of treating a variety of conditions, including conditions mediated by T cells, e.g. conditions mediated by memory T cells, e.g., skin conditions such as psoriasis, atopic dermatitis, T-cell lymphoma. skin, irritant and allergic contact dermatitis, lichen planus, alopecia areata, skin gangrene, vitiligo, ocular scar pemphigoid, UV damage and urticaria. The methods described herein concern the administration of multiple cycles of an inhibitor of the LFA-3 / CD2 interaction, for example, a soluble LFA-3 polypeptide, for example an LFA-3-Immunoglobulin (Ig) fusion protein. such as AMEVIVE® (Alefacept) (hereinafter AMEVIVE). It has been found that multiple courses of treatment with such agents provide more significant results (e.g., remarkably longer periods of remission) than a single therapy cycle or a double therapy cycle with, surprisingly, no apparent additional risk of side effects. In a preferred embodiment, the methods described herein concern the treatment of psoriasis. Accordingly, in one aspect, the invention features a method of treating a condition, for example, a skin condition such as psoriasis or other skin condition described herein. In one embodiment, the condition is mediated by memory effector T cells. The method includes administering a multiple course of treatment (preferably at least three cycles of treatment) of a soluble, bound to CD2-binding LFA-3 polypeptide to a subject. Preferably, the LFA-3 polypeptide that binds to CD2 is an LFA-3 fusion protein, for example, an LFA-3 / immunoglobulin (Ig) fusion protein. An exemplary LFA-3 / lg fusion protein includes an LFA-3 polypeptide that binds to CD2, soluble fused to all or part of a Fe region of an IgG, eg, fused to all or part of a joint region of heavy IgG chain and all or part of a heavy chain constant region. In a preferred embodiment, the Ig fusion protein consists of 92 amino-terminal amino acids of mature LFA-3, the C-terminal 10 amino acids of a human IgG1 articulation region, a CH2 region of a human IgGl heavy chain, t all or part of a CH3 region of a human IgG1 heavy chain. Such a fusion protein is AMEVIVE. AMEVIVE is encoded by an insert contained in plasmid pSAB152, deposited with American Type Culture Collection under accession number ATCC 68720. AMEVIVE is described in more detail later herein. The multiple course of treatment includes at least three treatment cycles, each cycle including (a) a period of administration during which a therapeutic agent is administered at least twice, followed by (b) a rest period during which the The therapeutic agent is not administered, wherein the rest period is substantially longer than the interval between administrations (IA) in the cycle, and preferably at least as long as the period of administration. In some modalities, the multiple run includes at least four cycles, five cycles, six cycles, seven cycles, eight cycles, nine cycles, ten cycles, eleven cycles of treatment or more. The period of administration of each cycle of the multiple run may be pre-selected or determined by, for example, a health care provider for the particular patient. Typically, the period of administration is sufficiently long to elicit a therapeutic response, for example, to elicit a selected level of remission which is determined by means of a clinical measurement such as the PASI evaluation. In some embodiments, the administration period is at least 8 weeks, at least 10 weeks, at least 12 weeks, at least 14 weeks, at least 20 weeks or more, but typically between 4 and 24 weeks . In a preferred embodiment, each cycle consists of 12 weeks of once-weekly administration of the polypeptide followed by 12 weeks of rest during which the patient is evaluated at least once for an effect of the agent, eg, a therapeutic effect or an effect secondary. In a preferred embodiment, the rest period of each successive cycle of the multiple run is longer than the rest period of a previous run in the multiple run. In some modalities, the rest period of the last cycle of the multiple run may be at least 2 years, of at least 18 months, of at least 3 years, of at least 4 years, 5 years or longer. The CD2-binding LFA-3 polypeptide can be administered in a dosage ranging from about 0.001 to about 50 mg of binding agent per kg body weight. In one embodiment, the polypeptide is administered in a generalized manner, preferably by the intramuscular (IM) or intravenous (IV) route. The period of administration typically includes periodic administration of the polypeptide, for example, once a week, twice a week, semi-weekly, or monthly. The polypeptide is typically administered in a unit dosage ranging from 2 to 15 mg when administered by the IV route (e.g., 7.5 mg bolus) and a unit dosage ranging from 2 to 30 mg when administered by the IM route (per example, IM injection of 10 or 15 mg). In one embodiment, the method includes evaluating the subject for the effects of the soluble CD2-binding LFA-3 polypeptide during one or both of the administration period and the rest period of each cycle in the multiple run. In one embodiment, the method includes administering to the subject an additional prophylactic or therapeutic agent during the multiple course of treatment, eg, UV radiation (e.g., UV radiation b) cyclosporin A, prednisone, FK506, methotrexate, steroids , retinoids, interferon and nitrogen mustard.

The additional agent can be administered during the administration period, during the rest period, or both, during one or more cycles. The subject is preferably a human. Preferred subjects include those that have symptoms of a T-cell mediated skin disorder such as psoriasis, eg, skin cell proliferation, elevated red plaque formation, scaly, pruritus, cracking, itching, burns or bleeding plaques, and those who have been diagnosed with psoriasis. In another aspect, the invention features methods of treating a subject who has psoriasis. The method includes (a) selecting a subject who has had at least two cycles, for example, selecting a subject on the basis of having had at least two cycles, of treatment with a soluble CD2-binding LFA-3 polypeptide. , and (b) administering an additional cycle, for example, a third, fourth, fifth, sixth, seventh, eighth, ninth, tenth or more cycles of treatment of an LFA-3 polypeptide binding to CD2 to the subject. In another aspect, the invention features methods of treating a subject, or of advising or advising a subject to be treated, with a CD2-binding LFA-3 polypeptide described herein. The method includes instructing or providing instructions to a subject, or to another individual, for example, to a health care provider, eg, a physician, nurse, hospital employee, HMO or other entity providing medical care, that a multiple run of the treatment described herein can be administered to the subject. The methods described herein can be used to treat any condition mediated by memory effector T cells. The methods described herein can be used to treat skin conditions such as psoriasis and scleroderma, and non-skin conditions such as inflammatory bowel diseases, psoriatic arthritis, rheumatoid arthritis, multiple sclerosis, and scleroderma.

BRIEF DESCRIPTION OF THE FIGURES Figures IA and IB present the nucleotide and amino acid sequences of an LFA-3 / lgG fusion protein. The signal peptide corresponds to amino acids 1-28 of Figure Ia; the mature LFA-3 region corresponds to amino acids 29-120 of Figure Ia; and the IgG1 region corresponds to amino acids 121-347 of Figure IA. Figure 2 is a bar graph of the percentage of psoriasis patients who achieved PASI 50 at 2 or 12 weeks after treatment for the A-D cycles of a multiple course of treatment with AMEVIVE. Figure 3 is a graph of the benefit and repetition of response in a multiple course of treatment with AMEVIVE for psoriasis. Figure 4 is a bar graph of the maximum length of response time in four psoriasis patients who received a multiple course of treatment with AMEVIVE. Figure 5 is a graph of mean CD4 + T cell counts for patients who have a multiple course of treatment with AMEVIVE. Figure 6A is a graph of the percentage of patients who achieved PASI 75 at any time of a multiple course of intravenous (IV) treatment with AMEVIVE. Figure 6B is a graph of the percentage of patients who achieved PASI 50 at any time of a multiple course of IV treatment with AMEVIVE. Figure 7A is a graph of the percentage of patients who achieved "clear" or "almost clear" PGA responses at any time of a multiple IV treatment run with AMEVIVE. Figure 7B is a graph of the percentage of patients who achieved "clear" or "almost clear" PGA responses at any time of a multiple run of IM treatment with AMEVIVE.

DETAILED DESCRIPTION OF THE INVENTION The methods described herein generally concern multiple run therapy with a soluble CD2-binding LFA-3 polypeptide., for the treatment of disorders disturbed by T cells (for example psoriasis). It was found that multiple-run therapy provides longer periods of remission than a single or dual cycle of therapy with, surprisingly, no apparent additional risk of side effects. Multiple Run Treatment As used herein, a "cycle" of treatment includes (a) a period of administration during which a therapeutic agent is administered at least twice.

(The interval between administrations is mentioned as IA), followed by (b) a rest period during which the therapeutic agent is not administered. The rest period is substantially longer, for example, at least 4-5 times longer, than the maximum AI, and is preferably at least as long as the administration period. During the period of administration of the cycle, the agent can be administered at least 2, 4, 6, 8, 10, 12, 14, 16, 18 or 20 times at (intervals (preferably regular). Typically, the administration period is sufficiently long for a patient to exhibit a pre-selected level of disease improvement, for example, a pre-selected PASI evaluation, for example, PASI 50 or PASI 75. The rest period may include monitoring the patient for a response to the therapeutic agent (eg, a therapeutic effect or a side effect) In a preferred cycle, the agent is administered once a week for a 12 week administration period followed by a 12 week rest period during the which the patient is evaluated by a health care provider at least once In the preferred modality there will be less than 50, 40, 30, 20 or 15 administrations during the administration period. Preferred Ades The largest interval between any two adjacent administrations (IA) in the cycle administration period is less than 30, less than 20, less than 15 or less than 10 days. In preferred embodiments, the interval between administrations is approximately one week. The period of administration of the cycle may vary with respect to the dosage strategy. For example, if the administration period is measured in weeks or months, the administration period may include monthly, weekly, bi-weekly, semi-weekly or daily administration of the agent for a specific number of weeks, as determined by a medical practitioner. for a particular patient. A preferred administration period of a cycle includes approximately 6-24 administrations with an AI of 3-15 days. More preferably, the period of administration of a cycle includes approximately 10-14 administrations with an AI of 5-9 days. In some cases the rest period is as much or longer than the period during which the agent has a substantial remitting effect on the patient, which was measured by a standard clinical measurement. For example, the rest period for a psoriasis patient during a treatment cycle may be the period during which a specific evaluation of the Severity and Psoriasis Area Index (PASI) (for example, PASI 50 or PASI 75) or a General Medical Evaluation (PGA) (PGA) (for example, "clear" or "almost clear" LGA) is maintained, or longer. However, a rest period that is at least as long as the remission period is typically between 1 and 10 years, for example, between 2 and 10 years, for example, between 2 and 5 years. In some modalities, the rest period is at least 1 year, preferably at least 18 months, 2 years, 30 months, 36 months, 48 months or longer. A "multiple treatment run" means at least three treatment cycles. The cycles in a multiple treatment run may be identical but not necessarily identical, for example, they may be different in the dosing strategy during the administration period; or in duration of either the IA, extension of the administration period, rest period, or both. For example, a multiple course of treatment may include (a) a first cycle consisting of 12 weeks of administration once weekly.

AMEVIVE followed by 12 weeks of rest, followed by (b) a second cycle consisting of 12 weeks of administration of AMEVIVE once weekly followed by a one-year rest period during which the agent has a substantial remitting effect, followed by (c) a third cycle consisting of 10 weeks of semi-weekly administration of the therapeutic agent followed by two years of rest, optionally followed by (d) successive cycles, eg, four, five, six, seven, eight or more additional cycles. In preferred embodiments, the rest period and the remitting effect increase with the increasing number of cycles during a multiple treatment run. The increase in the duration of the rest period or the remitting effect with each increasing cycle of treatment is preferably at least 10%, more preferably at least 15% or 20%, more preferably at least 25%, 30%, 40 %, 50% or more. In some modalities, the rest period and the remitting effect of the third cycle (and / or subsequent cycles) in a multiple course of treatment is at least 18 months, preferably at least 2 years, more preferably at least 30 months, 36 months, 42 months, 48 months or more. Inhibitors of the Interaction of CD2.LFA-3 Any inhibitor of the interaction of CD2: LFA-3 is useful in the methods of this invention. Such inhibitors include soluble LFA-3 polypeptides, anti-LFA-3 antibody homologs, anti-CD2 antibody homologs, soluble CD2 polypeptides, small molecules (eg, a chemical agent having a molecular weight of less than 2500 Da , preferably, less than 1500 Da, a chemical, for example, a small organic molecule, for example a product of a combinatorial library), LFA-3 and CD2 mimetics and derivatives thereof. Preferred inhibitors for use in the methods described herein are soluble CD2-binding LFA-3 polypeptides.

Soluble LFA-3 and CD2 Polypeptides Soluble LFA-3 polypeptides and soluble CD2 polypeptides that inhibit the interaction of LFA-3 and CD2 are useful in the methods of the present invention. Soluble LFA-3 polypeptides, in particular soluble Ig / LFA-3 fusions are preferred. As used herein, a "soluble CD2-binding LFA-3 polypeptide is a polypeptide that includes at least the CD2 binding domain of LFA-3 (SEQ ID NO: 2) and is incapable of anchoring itself Such soluble polypeptides include, for example, LFA-3 polypeptides that lack a sufficient portion of their membrane spacing domain to anchor the polypeptide or are modified so that the membrane spacing domain is not functional The soluble CD2-binding LFA-3 polypeptides include soluble fusion proteins that include at least the CD2 binding domain of LFA-3 fused to a heterologous polypeptide In one embodiment, the heterologous polypeptide is a Fe region of a immunoglobulin (e.g., an IgG1 linkage region and CH2-CH3 domains) or a substantial portion thereof Soluble LFA-3 polypeptides can be derived from the transmembrane form of LFA-3, particularly the extracellular minium (e.g., amino acids 1-187 of SEQ ID NO: 2 of U.S. 6,162,432, which is incorporated herein by reference). Such polypeptides are described in U.S. Pat. No. 4,956,281 and No. 6,162,432, which are incorporated herein by reference. Preferred soluble LFA-3 polypeptides include polypeptides that include residues 1-92 of SEQ ID NO: 2, residues 1-80 of SEQ ID NO: 2, residues 50-65 of SEQ ID NO: 2 and residues 20-80 of SEQ ID NO: 2, wherein SEQ ID NO: 2 are shown in US Pat. No. 6,162,432. A vector comprising a DNA sequence (SEQ ID NO: 1) encoding the amino acid sequence of SEQ ID NO: 2 is deposited with the American Type Culture Collection, Rockville, Maryland under Accession No. 75107, where SEQ ID NO: 1 and 2 are shown in US 6,162,432. The soluble LFA-3 polypeptides can also be derived from the Pl-linked form of LFA-3, such as those described in PCT Patent Application Serial No. WO 90/02181. A vector comprising a DNA sequence (SEQ ID NO: 3) encoding Pl-bound LFA-3 is deposited with the American Type Culture Collection, Rockville, Maryland under Accession No. 68788. It is understood that the form linked to Pl of LFA-3 and the transmembrane form of LFA-3 have identical amino acid sequences through the complete extracellular domain.

Accordingly, the preferred Pl-linked LFA-3 polypeptides are the same as for the transmembrane form of LFA-3. The most preferred soluble CD2-binding LFA-3 polypeptides for use in the present invention are LFA-3 / lg fusion proteins. An example of a fusion protein such AMEVIVE® (Alefacept). AMEVIVE® (Alefacept) AMEVIVE is a fusion protein that includes the first extracellular domain of human LFA-3 (CD58) fused to a Fe portion of human IgGl. In particular, AMEVIVE includes the 92 terminal amino acids of mature LFA-3, the 10 C-terminal amino acids of a human IgG1 joint region that contains the two cysteine residues although they participate in the interchain disulfide bond, and a substantial portion of the CH2 and CH3 of a human IgGl heavy chain constant domain (eg, SEQ ID NO: 8). The protein is a disulfide-linked, glycosylated dimer with a molecular weight of approximately 112 kD under non-reducing PAGE conditions. The AMEVIVE constant region has C-terminal variability corresponding to a spliced variant form of the full length fusion polypeptide. A plasmid, pSAB152, encoding AMEVIVE is deposited with American Type Culture Collection, Rockville, Maryland, under ATCC accession number 68720. pMDR (92) Ig-3 is an example of an expression vector that can be used to produce AMEVIVE pMDR (92) Ig-3 includes the following elements: (a) A segment of pBR322 that contains the ColEl origin and the "cassette" of beta lactamase expression (Access to GenBank No. J01749); (b) the DHFR expression cassette consisting of: SV40 early promoter with the enhancer removed (a portion of GenBank Accession No. J02400), rodent DHFR cDNA (Accession No. to GenBank L26316) , SV40 Poly A site, and the small intron t (portions of GenBank Accession No. J02400), and human gastrin transcription terminator sequence, 3'UTR (Sato et al. (1986) Mol. Cell Biol. 6: 1032-1043); (c) an AMEVIVE expression cassette that preferably includes in the following order: The early enhancer / promoter of SV40 (GenBank Accession No. J02400), Late Adenovirus Main Promoter and tripartite leader, which includes a donor splice and an intron sequence (a portion of Accession No. to GenBank J01917), Ig heavy chain variable region intron sequence and splice receptor (Kaufman and Sharp (1982) Mol Cell Biol. 2: 1304-1319, ( optionally) cloning linkers, the first 92 amino acids of the LFA-3 gene as isolated from a human tonsil cDNA library, fused in structure to a nucleic acid encoding the CH2 and CH3 joint regions of an IgG1 gene as isolated from a human fibroblastic genomic DNA library, cloning linkers (optionally), MIS 3'UT region that includes the poly site A (GenBank Accession No. K03474), and SV40 Poly A Site and Small Intron T (GenBank Accession No. J02400); and a segment of pBR327 (Access No. to GenBank L08856). Host cell lines that can be used to produce AMEVIVE can be derived from CHO-DukX-Bl cells. In one embodiment, a DHFR (-) mutant of this cell line can be transfected with the vector pMDR (92) Ig-3, and DHFR (+) transformants can be cultured on selective medium (eg, containing 25 nM methotrexate (MTX)). Positive transformants can be subjected to increasing concentrations of MTX (for example, 50 nM), and colonies that produce high levels of AMEVIVE can then be selected. The production of AMEVIVE can be carried out as follows: CHO host cells are thawed, scaled up to a 2000 L culture, maintained in culture for 6-7 days with pH control and nutrient feeding (48 hours, 96 hours, and 120 hours), after which the conditioned medium is harvested through microfiltration. MTX is preferably present in the culture medium. AMEVIVE can be recovered from the conditioned medium by carrying out the following steps: (i) Protein A chromatography, (ii) ceramic hydroxyapatite chromatography, (iii) viral inactivation at low pH, (iv) hydrophobic interaction chromatography, (v) followed by concentration, diafiltration, viral filtration, and a second stage of concentration that produces the fusion product Another way of producing AMEVIVE for use in the methods of this invention is described in the US Patent Application. with provisional No. 07 / 770,967 commonly assigned. Generally, conditioned culture medium from CHO or COS7 cells transfected with pSAB152 were concentrated using the spiral cartridge system AMICON S1Y30 AMICON, Danvers, Massachusetts) and subjected to chromatography on Protein A-Sepharose 4B (Sigma, St. Louis, Missouri). The bound proteins were eluted and subjected to Sepharose-12 gel filtration chromatography (Pharmacia / LKB, Picataway, New Jersey). The Sepharose-12 fractions containing AMEVIVE with the minimum amount of contaminating proteins, which was determined by SDS-PAGE gels and by Western staining analysis, (see, for example, Towbin et al., Proc.

Nati Acad. Sci. USA, 74 pp. 4350-54 (1979); Antibodies: A Laboratory Manual, pp. 474-510 (Cold Spring Harbor Laboratory (1988)), were combined and concentrated in a Centricon YM30 (AMICON). AMEVIVE was detected on Western stains using a polyclonal rabbit anti-LFA-3 antiserum, followed by detectably labeled goat anti-rabbit IgG. The AMEVIVE purified from COS7 and CHO cells was a dimer of two LFA-3-Ig fusion proteins, connected by disulfide bonds. The activity of the LFA-3-Ig fusion can be tested using the following bioassays: (1) a U937 CD32 / 64 bridging assay (Fe range of RI / RII), and (2) a cell bridging assay of Jurkat CD16 (Fe range of RUI). Both assays tested AMEVIVE's ability to bypass CHO cells that present cell surface CD2 to cells expressing Fc-gamma receptors. This assay, trial (2), involves culturing adherent CD2-CHO cells to form a monolayer in 96-well plates; add AMEVIVE controls and samples; add Jurkat-CD16 (+) cells fluorescently labeled; and measure the intensity of the fluorescence. Linkage of the fusion of LFA-3-Ig to CD2 immobilized on a substrate, eg, a microelement, can also be used to test the fusion proteins. CD2 polypeptides Soluble CD2 polypeptides can be derived from full length CD2, particularly the extracellular domain. Said polypeptides may comprise all or part of the extracellular domain of CD2. Exemplary soluble CD2 polypeptides are described in PCT WO 90/08187, which is incorporated herein by reference. Production of Soluble Polypeptides The production of the soluble polypeptides useful in this invention can be achieved by a variety of methods known in the art. For example, the polypeptides may be derived from intact transmembrane LFA-3 or from CD2 molecules or an intact Pl-bound LFA-3 molecule by proteolysis using specific endopeptidases in combination with exopeptidases, Edman degradation or both. The intact LFA-3 molecule or the intact CD2 molecule, in turn, can be purified from its natural source using conventional methods. Alternatively, intact LFA-3 or CD2 can be produced by known recombinant DNA techniques using cDNAs (see, for example, US Patent No. 4,956,281 to Wallner et al., Aruffo and Seed, Procc. Nati. Acad. Sci., 84 pp. 2941-45 (1987), Sayre et al, Proc. Nati, Acad. Sci. USA, 84 pp. 2941-45 (1987)). Preferably, the soluble polypeptides useful in the present invention are produced directly, thereby eliminating the need for an entire LFA-3 molecule or a whole CD2 molecule as an initial material. This can be achieved by conventional chemical synthesis techniques or by well-known recombinant DNA techniques wherein only those DNA sequences. which encode the desired peptides are expressed in transformed hosts. For example, a gene encoding the desired soluble LFA-3 polypeptide or soluble CD2 polypeptide can be synthesized by chemical means using an oligonucleotide synthesizer. Said oligonucleotides are designed based on the amino acid sequence of the desired soluble LFA-3 polypeptide or the soluble CD2 polypeptide. The specific DNA sequences encoding the desired peptide can also be derived from the full length DNA sequence by isolation of specific restriction endonuclease fragments or by PCR synthesis of the specific region. Standard methods can be applied to synthesize a gene encoding a soluble LFA-3 polypeptide or a polypeptide. of soluble CD2 that is useful in this invention. For example, the entire amino acid sequence can be used to construct a retro-translated gene. A DNA oligomer containing a nucleotide sequence encoding a soluble LFA-3 polypeptide or for a soluble CD2 polypeptide useful in this invention can be synthesized in a single step. Alternatively, several smaller oligonucleotides coding for portions of the desired polypeptide can be synthesized and then ligated. Preferably, a soluble LFA-3 polypeptide or a soluble CD2 polypeptide useful in this invention will be synthesized as several separate oligonucleotides that are subsequently ligated together. Individual oligonucleotides typically contain 5 'or 3' suspensions for complementary assembly. Once assembled, the preferred genes will be characterized by sequences that are recognized by restriction endonucleases (which include unique restriction sites for direct assembly into a cloning or expression vector) preferred codons that take into consideration the expression system of the host to be used, and a sequence that, when transcribed, produces an efficiently translated, stable mRNA. The appropriate assembly can be confirmed by formation of nucleotide sequences, restriction mapping and expression of a biologically active polypeptide in a suitable host.

Those skilled in the art will appreciate that due to the degeneracy of the genetic code, DNA molecules comprising many other nucleotide sequences will also be able to encode soluble CD2 and LFA-3 polypeptides encoded by the specific DNA sequences described above. . These sequences also degenerate the code for polypeptides that are useful in this invention. The DNA sequences can be expressed in unicellular hosts, or preferably in isolated mammalian host cells. As is well known in the art, in order to obtain high levels of expression of a transfected gene in a host, the gene must be operably linked to translational or transcriptional expression control sequences that are functional in the selected expression host. Preferably, the expression control sequences, and the gene of interest, will be contained in the expression vector which additionally comprises a bacterial selection marker and origin of replication. If the expression host is a eukaryotic cell, the expression vector may additionally comprise an additional expression marker useful in the expression host. The DNA sequences encoding the desired soluble polypeptides may or may not encode a signal sequence. If the prokaryotic expression host, it is generally preferred that the DNA sequence does not encode a signal sequence. If the expression host is eukaryotic, it is generally preferred that a signal sequence be encoded. An amino terminal methionine may or may not be present in the expressed product. If the terminal methionine is not fragmented by the expression host, it can, if desired, be chemically removed by standard techniques. A wide variety of host / vector combinations can be employed. Useful expression vectors for eukaryotic hosts, for example, vectors comprising expression control sequences from SV40, bovine papilloma virus, adenovirus and cytomegalovirus. Useful expression vectors for bacterial hosts include known bacterial plasmids, such as E. coli plasmids including E. coli, pCRl, pBR322, pMB9 and their derivatives, wider range of host plasmids, such as RP4, phage DNAs, by example the numerous lambda phage derivatives, for example, NM989, and other phage DNAs, such as M13 and phage and single-stranded filamentous phage DNA. Useful expression vectors for yeast cells include the 2μ plasmid and derivatives thereof. Useful vectors for insect cells include pVL 941. In addition, any of a wide variety of expression control sequences can be used in these vectors. Such useful expression control sequences include the expression control sequences associated with structural genes of the foreign expression vectors. Examples of useful expression control sequences include, for example, the late and early promoters of SV40 or adenovirus, the lac system, the trp system, the TAC or TRC system, the principal operator and the promoter regions of lambda phage, the regions of control of the fd coating protein, the promoter for 3-phosphoglycerate kinase or other glycolytic enzymes, the acid phosphatase promoters, e.g., Pho5, the promoters of the a-coupled yeast system and other known sequences for controlling expression of genes from prokaryotic or eukaryotic cells or their viruses, and various combinations thereof. A wide variety of host cells are useful. The host cells can be a unicellular organism, or can be obtained from multicellular organisms, for example, cells isolated from a multicellular host. These hosts may include well-known prokaryotic and eukaryotic hosts, such as strains of E. coli, Pseudomonas, Bacillus., Streptomyces, fungi, yeasts, insect cells such as Spodoptera frugiperda (SF9), animal cells such as CHO and mouse cells, African green monkey cells such as COSÍ, COS7, BSC1, BSC40 and BMT 10 cells, and human cells , as well as plant cells in tissue culture. For the expression of animal cells, CHO cells and COS 7 cells are preferred. It will be understood that not all vectors and expression control sequences will work equally well to express the DNA sequences described herein. Not all guests will function equally well with the same system of expression. However, a person skilled in the art can make a selection between these vectors, expression control sequences and hosts without undue experimentation. For example, when selecting a vector, the host must be considered because the vector must replicate in it. The number of copies of the vector, the ability to control that number of copies, and the expression of any other of the proteins encoded by the vector, such as antibiotic markers, should also be considered. When selecting an expression control sequence, a variety of factors should also be considered. These include, for example, the relative strength of the sequence, its controllability, and its compatibility with the DNA sequences discussed herein, particularly with respect to potential secondary structures. Single-celled hosts should be selected in consideration of their compatibility with the selected vector, the toxicity of the product encoded by the DNA sequences, their secretion characteristics, their ability to correctly bend the soluble polypeptides, their fermentation or culture requirements, and the ease of purification of the products encoded by the DNA sequences. In these parameters, one of skill in the art can select various combinations of host / exprεε / vector control sequence which will express the desired DNA sequences in fermentation or in large scale animal culture, for example with CHO cells or COS 7 cells. The soluble CD2 and LFA-3 polypeptides can be isolated from fermentation or cell culture and purified using any of a variety of conventional methods. A person skilled in the art can select the appropriate purification and isolation techniques. Although recombinant DNA techniques are the preferred method of producing useful soluble CD2 polypeptides or soluble LFA-3 polypeptides having a sequence of more than 20 amino acids, shorter LFA-3 or CD2 polypeptides having less than about 20 amino acids are preferably produced by means of conventional chemical synthesis techniques. The synthetically produced polypeptides useful in this invention can be advantageously produced in extremely high yields and can be easily purified. Preferably, said soluble CD2 polypeptides or soluble LFA-3 polypeptides are synthesized by solid phase or solution phase polypeptide synthesis and, optionally, are digested with carboxypeptidase (to eliminate C-terminal amino acids) or degraded by of Ed a manual degradation (to eliminate the N-terminal amino acids). The use of phase synthesis in solution allows the direct addition of certain derivatized amino acids to develop the polypeptide chain, such as tyrosine O-sulfate ester. This obviates the need for a subsequent derivatization step to modify any residues of the polypeptides useful in this invention. Proper dubbing of the polypeptides can be achieved under oxidative conditions that favor disulfide bridge formation as described in Kent, "Chemical Synthesis of Polypeptides and Proteins", Ann. Rev. Biochem., 57, pp. 957-89 (1988). The polypeptides produced in this way can then be purified by means of separation techniques well known in the art. Homologues of Anti-CD2 and Anti-LFA-3 Antibodies As used herein, an "antibody homologue" is a protein comprising one or more polypeptides selected from immunoglobulin light chains, heavy immunoglobulin chains and antigenic binding fragments thereof, which are capable of binding to an antigen. The polypeptide components of an antibody homologue composed of more than one polypeptide can optionally be linked to a disulfide otherwise covalently crosslinked. Accordingly, antibody homologs include intact immunoglobulins of the IgA, IgG types; IgE, IgD, IgM (as well as subtypes thereof), wherein the immunoglobulin light chains can be of the kapa or lambda types. Antibody homologs also include portions of intact immunoglobulins that retain the antigenic binding specificity, eg, Fab fragments, Fab 'fragments, fragments (F (ab') 2, F (v) fragments, monomers or heavy chain dimers, monomers or dimers of light chain, dimers consisting of a heavy chain and a light chain, and the like.The term includes recombinant antibodies, antibodies humanized and grafted to CDR, chimeric, or other antibodies modified to be less immunogenic in a human As herein, a "humanized or recombinant humanized antibody homologue" is an antibody homolog, produced by recombinant DNA technology, in which some or all of the amino acids of a human immunoglobulin light or heavy chain that is require for antigenic binding have been replaced by corresponding amino acids from a heavy or light immunoglobulin chain of m non-human amide. As herein, a "chimeric recombinant antibody homolog" is an antibody homolog, produced by recombinant DNA technology, in which all or part of the constant and articulation regions of a heavy chain, immunoglobulin light chain , or both, have been replaced by the corresponding regions from another heavy chain or immunoglobulin light chain. Many types of homologues of anti-CD2 or anti-LFA-3 antibodies are ul in the methods of this invention. These include monoclonal antibodies, recombinant antibodies, chimeric recombinant antibodies, humanized recombinant antibodies, as well as antigenic binding portions of the foregoing. Among anti-LFA-3 antibody homologs, it is preferable to monoclonal anti-LFA-3 antibodies. It is more preferable to a monoclonal anti-LFA-3 antibody produced by a hybridoma selected from the group of hybridomas having ATCC Accession Nos. HB 10693 (IE6), ATCC HB 10694 (HC-lBll), ATCC HB 10695 (7A6), and ATCC HB 10696 (8B8), or the monoclonal antibody known as TS2 / 9 (Sánchez-Madrid et al., "Three Distinct Antigens Associated with Human T-Lymphocyte-Mediated Cytolysis: LFA-1, LFA-2 and LFA-3" , Proc. Nati, Acad. Sci. USA, 79, pp. 7489-93 (1982)). More preferably, the monoclonal anti-LFA-3 antibody is produced by a hybridoma selected from the group of hybridomas having ATCC Accession Nos. HB 10695 (7A6) and ATCC HB 10693 (IE6). Among anti-CD2 antibody homologs, it is preferable to monoclonal anti-CD2 antibodies, such as the anti-CD2 monoclonal antibodies known as Tlla epitope antibodies, which include TS2 / 18 (Sánchez-Madrid et al., "Three Distinct Antigens Associated with Human T-Lymphocyte-Mediated Cytolysis: LFA-1, LFA-2 and LFA-3", Proc. Nati, Acad. Sci. USA, 79, pp. 7489-93 (1982).) The technology to produce antibodies Monoclonal antibodies are well known, see generally, Harlow, E. and Lane, D. (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor, Laboratory Press, Cold Spring Harbor, NY; Kohier et al., nature, "Continuous Cultures of Fused Cells Secreting Antibody of Predefined Specificity, 256 pp, 495-97 pp. 495-497 (1975). Immunogens useful for the purpose of this invention include cells possessing LFA-3 or CD2, as well as cell-free preparations that they contain LFA-3, CD2 or binding fragments of the counter-receptor thereof (eg, fragments of CD2 that bind to LFA-3 or fragments of LFA-3 that bind to CD2.) Immunization can be achieved using standard procedures. unit dose and the immunization regimen depend on mammalian species immunizes two, their immune status, the body weight of the mammal, etc. Typically, the immunized mammals are bled and the serum from each of the blood samples is assayed for particular antibodies using screening assays. For example, anti-CD2 or anti-LFA-3 antibodies can be identified by testing the ability of the immune serum to block red blood cells from sheep that emerge from Jurkat cells, which results from the presence of LFA -3 and CD2 on the respective surfaces of these cells. The lymphocytes used in the production of hybridoma cells are typically asylated from immunized mammals whose serum has already proven to be positive for the presence of the desired antibodies using such screening assays. The homologues of anti-CD2 and anti-LFA-3 antibodies useful in the present invention can also be recombinant antibodies produced by host cells transformed with DNA encoding the immunoglobulin heavy and light chains of a desired antibody. Recombinant antibodies can be produced by well-known genetic engineering techniques. See, for example, U.S. Pat. No. 4,816,397, which is incorporated herein by reference. For example, recombinant antibodies can be produced by cloning cDNA or genomic DNA encoding the immunoglobulin heavy and light chains of the desired antibody from a hybridoma cell that produces an antibody homolog useful in this invention. The cDNA or genomic DNA encoding the polypeptides is then inserted into expression vectors so that both genes are operably linked to their own translational and transcriptional expression control sequences. The expression vector and the expression control sequences are selected to be compatible with the expression host cell used. Typically, both genes are inserted into the same expression vector. Prokaryotic or eukaryotic host cells can be used. Expression in eukaryotic host cells is preferred because said cells are more likely than prokaryotic cells to conjugate and secrete an immunologically active antibody and properly bent. It is possible that the host cells will produce portions of isolated antibodies, such as light chain dimers or heavy chain dimers, which are also antibody homologs according to the present invention. It will be understood that variations on the above procedure are useful in the present invention. For example, it may be desired to transform a host cell with DNA encoding either the light chain or the heavy chain (but not both) of an antibody homologue. Recombinant DNA technology can be used to remove some or all of the DNA encoding either or both or both of the heavy and light chains that is not necessary to bind to the CD2 or LFA-3 receptor counter. Molecules expressed from such truncated DNA molecules are useful in the methods of this invention. further, bifunctional antibodies may be produced in which a heavy and a light chain are homologous with anti-CD2 or anti-LFA-3 antibodies and the other light and heavy chain are specific for an antigen other than CD2 or LFA-3, or other Epitope of CD2 or LFA-3. Homologs of chimeric recombinant anti-CD2 or anti-LFA-3 antibodies can be produced by transformation of a host cell with a suitable expression vector comprising DNA encoding the desired heavy and light immunoglobulin chains in which all or something of the DNA encoding the constant and articulation regions of the light chain and / or the heavy chain have been replaced with DNA from the corresponding region of a heavy or light immunoglobulin chain of a different species. When the original recombinant antibody is not human, and the inhibitor is to be administered to a human, substitution of the corresponding human sequences is preferred. An exemplary chimeric recombinant antibody has mouse variable regions and human constant and articulation regions. See generally, U.S. Pat. No. 4,816,397; Morrison et al., "Chimeric Human Antibody Molecules: Mouse Antigen-Binding Domains With Human Constant Region Domains", Proc. Nati Acad. Sci. Usa, 81, pp. 6851-55 (1984); Robinson et al., International Patent Publication PCT / US 86/02269; Akira et al., European Patent Application 184,187; Tamiguchi, M., European Patent Application 171,496; Neuberger et al., International Application WO 86/01533; Better et al. (1988 Science 240: 1041.1043); Liu et al. (1987) PNAS 84: 3439-3443; Liu et al., 1987, J. Immunol. 139: 3521-3526; Sun et al. (1987) PNAS 84: 214-218; Nishimura et al., 1987, Canc. Res. 47: 999-1005; Wood et al (1985) Nature 314: 446-449; and Shaw et al., 1988, J. Nati. Cancer Inst. 80: 1553-1559. Humanized recombinant anti-CD2 or anti-LFA-3 antibodies can be generated by replacing the variable region Fv sequences that are not directly involved in the antigenic binding with equivalent sequences of the human Fv variable regions. General methods for generating humanized antibodies are provided in Morrison, S.L., 1985, US 5,585,089, US 5,693,761 and US 5,693,762, the total contents of which are incorporated herein by reference. These methods include isolating, manipulating and expressing nucleic acid sequences encoding all or part of the immunoglobulin Fv variable regions. The sources of such nucleic acids are well known to those skilled in the art and, for example, can be obtained from the hybridoma by producing an anti-LFA-3 or anti-CD2 antibody. The nucleic acids encoding a humanized antibody, or fragment thereof, can then be cloned into an appropriate expression vector.

Molecules of CDR-grafted antibody or immunoglobulins can be produced by CDR grafting or by substitution of CDR, wherein one, two, or all CDRs of an immunoglobulin chain can be replaced. See, for example, US Patent 5,225,539; Jones et al. 1986, Nature 321: 552-525; Verhoeyan et al 1988 Science 239: 1534; Beidler et al 1988 J. Immunol. 141: 4053-4060; Winter US 5,225,539, the contents of which are expressly incorporated herein by reference. Winter discloses a method of grafting CDR that can be used to prepare the humanized antibodies of the present invention (UK Patent Application GB 2188638A filed on March 26, 1987; Winter US 5,225,539), the contents of which are expressly incorporated by reference. All CDRs of a particular human antibody can be replaced with at least a portion of a non-human CDR or only some of the CDRs can be replaced with non-human CDRs. It is only necessary to replace the number of CDRs required for the binding of the humanized antibody to a predetermined antigen, for example, LFA-3 or CD2. Also within the scope of the invention are humanized antibodies, including immunoglobulins, in which specific amino acids have been substituted, deleted or added. In particular, preferred humanized antibodies have amino acid substitutions in the structural region, such as improving the binding to the antigen. For example, a small number of selected structural residues of the humanized immunoglobulin chain can be replaced by the corresponding donor amino acids. Preferred locations of the substitutions include amino acid residues adjacent to the CDR, which waves are capable of interacting with a CDR (see, for example, US 5,585,089). The criteria for selecting amino acids from donors is described in US 5,585,089, for example, columns 12-16 of US 5,585,089 whose contents are incorporated herein by reference. Other techniques for humanizing immunoglobulin chains, including antibodies, are described in Padlan et al. EP 519 596 Al, published on December 23, 1992. Human monoclonal antibodies (mAbs) directed against LFA-3 or CD2 can be generated using mice transgenic that carry the complete human immune system preferably to the mouse system. Splenocytes of these transgenic mice immunized with the antigen of interest are used to produce hybridomas that secrete human mAbs with specific affinities for epitopes of a human protein (see, for example, Wood et al. International Application WO 91/00906; Kucherlapati et al. PCT WO 91/10741; Lonberg et al. International Application WO 92/03918; Kay et al. International Application 92/03917; Lonberg N et al. 1994 Nature 368: 856-859; Green, LI et al. 1994 Nature Genet. -21; Morrison, SL et al 1994 Proc. Nati, Acad. Sci. USA 81: 6851-6855; Bruggeman et al 1993 Year Immunol., 7: 33-40; Tuaillon et al. 1993 PNAS 90: 3720-3724; Bruggeman et al. collaborators 1991 Eur J. Immunol 21: 1323-1326). Monoclonal antibodies can also be generated by other methods known to those skilled in the art of recombinant DNA technology. An alternative method, referred to as the "combinatorial antibody display" method, has been developed to identify and isolate antibody fragments that have a particular antigenic specificity, and can be used to produce monoclonal antibodies (for description of the combinatorial antibody display see, for example, Sustry et al 1989 PNAS 86: 5728; Huse et al 1989 Science 246: 1275; and Orlandi et al 1989 PNAS 86: 3833). After immunizing an animal with an immunogen as described above, the repertoire of antibodies from the resulting B cell pool is cloned. The methods are generally known to obtain the DNA sequence of the variable regions of a diverse population of immunoglobulin molecules using a mixture of oligomeric primers and PCR (Larrick et al., 1991, Biotechniques 11: 152-156; Larrick et al., 1991, Methods: Companion to Methods in Enzymology 2: 106-110). Examples of particularly manageable methods and reagents can be found for use in generating a library displaying the variegated antibody in, for example, Ladner et al. Patent US No. 5,223,409; Kang et al. International Publication No. WO 92/18619, Dower et al. International Publication No. WO 91/17271; Winter et al. International Publication No. WO 92/20791; Markland et al. International Publication No. WO 92/15679; Breitling et al. International Publication No. WO 93/01288; McCafferty et al. International Publication No. WO 92/01047; Garrard et al. International Publication No. WO 92/09690; Ladner et al. International Publication No. WO 90/02809; Puchs et al. (1991) Bio / Technology 9: 1370-1372; Hay and collaborators (1992) Hum Antibod Hybrido as 3: 81-85; Huse et al (1989) Science 246: 1275-1281; Griffths et al. (1993) EMBO J 12: 725-734; Hawkins et al. (1992) J Mol Biol 226: 889-896; Clackson et al. (1991) Nature 352: 624-628; Gram et al. (1992) PNAS 89: 3576-3580; Garrad et al. (1991) Bio / Technology 9: 1373-1377; Hoogenboom et al. (1991) Nuc Acid Res. 19: 4133-4137; and Barbas et al. (1991) PNAS 88: 7978-7982. Equipment for generating phage display libraries is commercially available (e.g., the Pharmacia Recombinant Phage Antibody System, Catalog No. 27-9400-01, and the Stratagene SurfZAP ™ phage display kit, Catalog No. 240612). In certain embodiments, the domains of the B region of heavy and light chains can be expressed on the same polypeptide, linked by a flexible linker to form a single chain Fv fragment and the scFV gene can subsequently be cloned into the expression vector or desired phage genome. As was generally described in McCafferty et al., Nature (1990) 348: 552-554, the complete VL and VH domains of an antibody, linked by a flexible linker (Gly4-Ser) 3 can be used to produce a single chain antibody which can convert the separable presentation package based on the antigenic affinity. The scFV antibodies isolated immunoreactive with the antigen can be formulated in a pharmaceutical preparation for use in the method in question.

Specific antibodies with high affinities for a surface protein can be made in accordance with methods known to those skilled in the art, for example, methods involving library screening (Ladner, RC et al., US Patent No. 5,233,409; Ladner, R. C, et al., US Patent No. 5,403,484). Additionally, the methods of these libraries can be used in screens to obtain binding determinants that are mimetic of the structural determinants of the antibodies. See, for example, Bajorath, J. and Sheriff, 1996, Proteins: Struct., Funct., And Genet. 24 (2), 152-157; Webster, D. M. and A. R. Rees, 1995, "Molecular modeling of antibody-combining sites", in S. Paul, Ed., Methods in Molecular Biol. 51, Antibody engineering Protocols, Human Press, Totowa, NJ, p. 17-49; and Johnson, G., Wu, T. T. and E. A. Kabat, 1995, "Seqhunt: A program to screen nucleotide and amino acid sequences", in methods in Molecular Biol. 51, op. cit., p. 1-15. Homologues of anti-CD2 and anti-LFA-3 antibodies that are not intact antibodies are also useful in this invention. Said homologs can be derived from any of the antibody homologs described above. For example, antigenic binding fragments, as well as monomeric, dimeric and full-length trimeric polypeptides derived from the aforementioned antibodies are themselves useful. Useful antibody homologs of this type include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F (ab ') 2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge in the articulated region; (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single branch of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341: 544-546), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR).

Furthermore, although the two domains of the Fv, VL and VH fragment are encoded by separate genes, they can be linked, using recombinant methods, by means of a synthetic linker that facilitates them to be elaborated as a single protein chain in which the VL and VH pair to form monovalent molecules (known as single chain Fv (scFv), see for example, Bird et al (1988) Science 242: 423-426; and Huston et al. (1988) Proc. Nati. Acad. Aci. USA 85: 5879-5883). Said single chain antibodies are also intended to be encompassed by the term "antigenic binding fragment" of an antibody. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for utility in the same manner as are the intact antibodies. Heavy chains of anti-LFA-3 are preferred from the anti-LFA-3 antibody fragments. Antibody fragments can also be produced by chemical methods, for example, by fragmentation of an intact antibody with a protease, such as pepsin or papain, and optionally treatment of the fragmented product with a reducing agent. Alternatively, useful fragments can be produced using host cells transformed with truncated light chain and / or heavy chain genes. The light chain and heavy chain monomers can be produced by treatment of an intact antibody with a reducing agent, such as dithiothreitol, followed by purification to separate the chains. The heavy and light chain monomers can also be produced by means of host cells transformed with DNA encoding either the desired heavy chain or light chain, but not both. See, for example, Ward et al., "Binding Activities of a Repertoire of Single Immunoglobulin Variable Domains Secreted from Escherichia coli", Nature, 341. p. 544-46 (1989); Sastry et al., "Cloning of Immunological Repertoire in Escherichia coli for Generation of Monoclonal Catalytic Antibodies: Construction of a Heavy Chain Variable Region-Specific cDNA Library", Proc. Nati Acad. Sci. USA 86, p. 5728-32 (1989). Small Molecule or Mimetic Agents of LFA-3 and CD-2 The mimetics of LFA-3 and CD2 are also useful in the methods of this invention. These agents, which may be peptides, semi-speci fi c compounds or non-peptidic compounds (e.g., small organic molecules), are inhibitors of the CD2: LFA-3 interaction. The preferred LFA-3 and CD2 mimetics will inhibit the interaction of CD2: LFA-3 at least as well as the anti-LFA-3 monoclonal antibody 7A6 or the anti-CD2 monoclonal antibody TS2 / 18 (described supra). In preferred embodiments, the test agent is an element of a combinatorial library, for example, an organic or peptide combinatorial library, or a library of natural products. In a preferred embodiment, the plurality of the test compounds, eg, elements of the library, includes at least 10, 102, 103, 104, 105, 10, 107, or 108 compounds. In a preferred embodiment, the plurality of test compounds, for example, elements of the library. they share a structural or functional characteristic. In one embodiment, the invention provides libraries of LFA-3 and / or CD2 inhibitors. The synthesis of combinatorial libraries is well known in the art and has been reviewed (see, for example, EM Gordon et al, J. Med. Chem. (1994) 37: 1385-1401; De Witt, SH; Czarnik, AW Acc Chem. Res. (1996) 29: 114; Armstrong, R.; Combs, AP; Te pest, PA; Brown, SD; Keating TA Acc. Chem. Res. (1996) 29: 123; Ellman, JA Acc Chem. Res. (1996) 29: 132; Gordon, EM; Gallop, MA; Patel, DV Acc. Chem. Res. (1996) 29: 144; Lowe, G. Chem. Soc. Rev. (1995) 309 , Blobdelle and colleagues Trends Anal, Chem. (1995) 14:83, Chen et al. J. Am. Chem. Soc. (1994) 116: 2661, US Patent Nos. 5,359,115, 5,362,899, and 5,288,514; PCT Publication Nos. WO 92/10092, WO 93/09668, WO 91/07087, WO 93/20242, WO 94/08051). The libraries of compounds of the invention can be prepared according to a variety of methods, some of which are known in the art. For example, a "subdivided set" strategy may be implemented in the following manner: beads of a functionalized polymeric support are placed in a plurality of reaction vessels; a variety of polymeric supports suitable for solid phase peptide synthesis are known, and some are commercially available (see, for example, M. Bodansky "Principies of Peptide Synthesis ", 2nd Edition, Springer-Verlag, Berlin (1993)). To each aliquot of beads is added a solution of a different activated amino acid, and the reactions are allowed to proceed to produce a plurality of immobilized amino acids, one in each reaction vessel. The aliquots of derivatized beads are then washed, "pooled" (i.e., recombined), and the bead set is again divided, with each aliquot being placed in a separate reaction vessel. Another activated amino acid is then added to each aliquot of beads. The synthesis cycle is repeated until a desired peptide extension is obtained. The amino acid residues added to each synthesis cycle can be randomly selected; alternatively, amino acids can be selected to provide a "biased" library, for example, a library in which certain portions of the inhibitor are selected non-randomly, for example, to provide an inhibitor having similarity or known structural homology to a known peptide capable of interacting with an antibody, for example, the anti-idiotypic antibody antigen binding site. It will be appreciated that a wide variety of peptide, peptidomimetic, or non-peptide compounds can be easily generated in this manner. The "subdivided set" strategy results in a library of peptides, for example, inhibitors, which can be used to prepare a library of test compounds of the invention. In another illustrative synthesis, a "diversity library" is created by the method of Hobbs DeWitt et al. (Procc.Nat.Academ.Sci.U.S.A. 90: 6909 (1993)). Other synthesis methods, including the "tea bag" technique of Houghten et al., Nature 354: 84-86 (1991)) can also be used to synthesize libraries of compounds according to the invention. The libraries of compounds can be screened to determine if some elements of the library have the desired activity, if they do, identify the active species. Methods of screening conventional libraries have been described (see, for example, Gordon et al., J. Med. Chem. Supra). Libraries of soluble compounds can be screened by affinity chromatography with the appropriate receptor to isolate ligands from the receptor, followed by identification of the isolated ligands by means of conventional techniques (e.g., mass spectrometry, NMR, and the like).

The immobilized compounds can be screened by contacting the compounds with a soluble receptor; preferably, the soluble receptor is conjugated to a tag (eg, colorimetric enzymes, radioisotopes, luminescence compounds, and the like) that can be detected to indicate binding of the ligand. Alternatively, the immobilized compounds can be selectively released and allowed to diffuse through a membrane to interact with a receptor. Exemplary assays useful for screening libraries of the invention are described below. In one embodiment, the compounds of the invention can be screened by the ability to interact with a CD2 or LFA-3 polypeptide by assaying the activity of each compound to bind directly to the polypeptide or to inhibit a CD2: LFA-3 interaction. , for example, by incubating the test compound with an LFA-3 or CD2 polypeptide and a lysate, for example, a T cell lysate or APC, for example, in a well or multi-well plate, such as the microtiter plate. of 96 standard wells. In one embodiment, the activity of each individual compound can be determined. A well or wells that do not have test compounds can be used as a control. After incubation, the activity of each test compound can be determined by assaying each well. Thus, the activity of a plurality of test compounds in parallel can be determined. In yet another embodiment, a large number of test compounds can be tested simultaneously for binding activity. For example, test compounds can be synthesized on solid resin beads in "one compound-one bead" synthesis; the compounds can be immobilized on the resin support by means of a photolabile linker. A plurality of beads (eg, as many as 100,000 beads or more) can then be combined with yeast cells and sprayed into a plurality of "nano-droplets", in which each droplet includes a single bead (and therefore, a single bead). test compound). The exposure of the nano-droplets to UV rays then results in the fragmentation of the compounds from the beads. It will be appreciated that these assays allow the screening of large libraries of test compounds in a rapid format. Combinatorial libraries of compounds with "tags" can be synthesized to encode the identity of each library element (see, eg, WC Still et al., US Patent No. 5,565,324 and PCT Publication Nos. WO 94/08051 and WO 95 / 28640). In general, this method is characterized by the use of inert, but easily detectable labels that are fixed to the solid support or to the compounds. When an active compound is detected (for example, by means of one of the techniques described above), the identity of the compound is determined by identification of the unique label that accompanies it. This marking method allows the synthesis of large libraries of compounds that can be identified at very low levels. Such a marking scheme may be useful, for example in the "nano-droplet" screening assay described above, to identify compounds released from the beads. In preferred embodiments, libraries of compounds of the invention contain at least 30 compounds, more preferably at least 100 compounds, and even more preferably at least 500 compounds. In preferred embodiments, libraries of compounds of the invention contain less than 109 compounds, more preferably less than 108 compounds, and even more preferably less than 107 compounds. DERIVATIZED INHIBITORS Also useful in the methods of this invention are inhibitors derivatized from the interaction of CD2: LFA-3 in which, for example, any of the antibody homologues, soluble LFA-3 and CD2 polypeptides, or LFA mimics. -3 and CD2 described herein are functionally linked (via chemical coupling), genetic fusion or otherwise) to one or more elements independently selected from the group consisting of anti-CD2 and anti-LFA-3 antibody homologues, soluble LFA-3 and CD2 polypeptides, LFA-3 and CD2 mimetics , cytotoxic agents and pharmaceutical agents. One type of derivatized inhibitor is produced by crosslinking two or more inhibitors (of the same type or of different types). Suitable crosslinkers include those which are heterobifunctional, having two reactive groups interchangeably separated by an appropriate spacer (e.g., m-maleimidobenzoyl-N-hydroxysuccinimide ester) or homobifunctional (e.g., disuccinimidyl suberate). Such spacers are available from Pierce Chemical Company, Rockford, Illinois. Another possibility of crosslinking takes advantage of the Pl signal sequence in LFA-3 bound to Pl, or fragments thereof. Specifically, the DNA encoding the Pl binding signal sequence (eg, amino acids 162-212 of SEQ ID NO: 4) is ligated from the 5 'end to the 3' end in the direction of the strand. of duplex DNA encoding a desired polypeptide, preferably a soluble LFA-3 polypeptide. If this construct is expressed in an appropriate eukaryotic cell, the cell will recognize the Pl binding signal sequence and covalently bind Pl to the polypeptide. The hydrophobic property of Pl can then be exploited to form micellar agglutinates of the polypeptides. Also useful are polypeptides linked to one or more pharmaceutical or cytotoxic agents. Useful pharmaceutical agents include biologically active peptides, polypeptides and proteins, such as antibody homologs specific for a human polypeptide other than CD2 or LFA-3, or portions thereof. Pharmaceutical agents and useful cytotoxic agents also include UV radiation, (eg, UV B), cyclosporin A, prednisone, FK506, methotrexate, steroids, retinoids, interferons, and nitrogen mustard. Preferred pharmaceutically-derivatized inhibitors include the recombinantly produced polypeptide in which a soluble LFA-3 polypeptide, soluble CD2 polypeptide, or a peptidyl CD2 or peptidyl LFA-3 mimetic is fused to all or part of a region of heavy immunoglobulin chain linkage to all or part of a heavy chain constant region. Preferred polypeptides for preparing said fusion proteins are soluble LFA-3 polypeptides. More preferred are fusion proteins containing amino acids 1-92 of mature LFA-3 fused to a portion of a human IgG1 linkage region (which includes the ten C-terminal amino acids of the hinge region containing two cysteine residues). even to participate in the interchain disulfide bond) and the CH2 and CH3 regions of a heavy chain constant domain of IgGl. It is expected that said fusion proteins inhibit the prolonged half-lives of serum and facilitate the dimerization of the inhibitor. The utility in the methods of this invention of specific soluble CD2 polypeptides, soluble LFA-3 polypeptides, anti-LFA-3 antibody homologs, anti-CD2 antibody homologues or LFA-3 and CD2 mimetics can be easily determined rehearsing its ability to inhibit the interaction of LFA-3 / CD2. This ability can be tested, for example using a simple cell binding assay that allows visual evaluation (under amplification) of the ability of the putative inhibitor to inhibit the interaction between LFA-3 and CD2 on cells that possess these molecules. Jurkat cells are preferred as the CD2 + substrate and sheep red blood cells or human JY cells are preferred as the LFA-3 + substrate. The binding characteristics of soluble polypeptides, antibody homologs and mimetics useful in this invention can be tested in a variety of preferred ways, such as radiolabelling the antibody, polypeptide or agent homologue (e.g., with 35S or 15I) and then putting in contact the labeled polypeptide, mimic agent or antibody homologue with CD2 + cells of LFA-3 +, as appropriate. The binding characteristics can also be assayed using an appropriately enzyme labeled secondary antibody. Rosette comparison assays such as those described by Seed et al. (Proc. Nati. Sci. USA, 84 pp. 3365-69 (1987)) may also be used. Combination Therapy Agents, for example, soluble CD2-binding LFA-3 polypeptides can be used in combination with other therapies, for example, other agents. The other agents are referred to herein as "second agents" or "additional agents" and include one or more of: an immunosuppressant (e.g., methotrexate, cyclosporine, or chlorambucil), cyclophosphamide, prednisone, FK506, steroids, retinoids, interferon , Nitrogen mustard, a cytokine binding agent (for type 2, for example an IL-2 or IL-8 binding agent, for example, an anti-IL-2 or anti-IL-8 monoclonal antibody (Abgenix )), an inhibitor of an ICAM / LFA-1 interaction, for example, an ICAN binding agent (eg, an antibody, eg, a monoclonal antibody) against ICAM-1 (eg, an anti-HIV antibody). Humanized, chic or human ICAM-1); or an LFA-1 binding agent (also known as CDlla) (e.g., an antibody, e.g., a monoclonal antibody) against LFA-1 (e.g., a humanized, chic or human anti-LFA-1 antibody, for example, Raptiva (Genentech / Xoma)); a binding agent to a co-stimulatory molecule, for example a binding agent to B7-1 (CD80) (for example, an anti-B7-l monoclonal antibody (IDEC); a vasodilator (for example, an ACE inhibitor or minoxidil), a corticosteroid or penicillamine In one embodiment, the agent, for example, an inhibitor of the interaction of CD2: LFA-3, is administered in combination with one or more inhibitors of interleukin-1 (IL-1), IL -2, IL-4, IL-6, IL-8, TGF-β, PDGF, granzi a A or leukotriene B 4. Such a combination therapy can advantageously use lower dosages of the therapeutic or prophylactic agents administered "in combination" , as used herein, means that two (or more) different treatments are released to the subject during the course of the affliction of the subject with the disorder, for example, the two or more treatments are released after the subject has been diagnosed with the disorder and before the disorder has been cur Adopted or deleted. In some modalities, the release of a treatment occurs even when the release of the second begins, so that there is an over position. This is sometimes referred to in the present as "concurrent release" or "simultaneous" release. In other modalities, the release of the treatment ends before the release of the other treatment begins. In some modalities of one case or another or both, the treatment is more effective because of the combined administration. For example, the second treatment is more effective, for example, an equivalent effect is seen with less than the second treatment, or the second treatment reduces symptoms to a greater degree, than would be seen if the second treatment was administered in the absence of first treatment, or the analogous situation is seen with the first treatment. In some modalities, the release is that the reduction of symptoms, or other parameters related to the disorder, for example reduction in the level of T cells or activity, is greater than what would be observed with a treatment released in the absence of the other. The effect of the two treatments may be partially additive, complete additive or greater than additive. The release may be such that an effect of the first treatment released is still detectable when the second is released, for example, when the binding agent to LFA-3 or CD2- is first released, a reduction in the level or activity of T cells. it is still detectable when the second agent is released. In a preferred embodiment, a release of the first treatment and a release of the second treatment takes place in 1, 2, 5, 10, 15 or 30 days from one another. In a preferred embodiment, the CD2 binding agent (eg, the LFA-3 / lg fusion), the second agent (or both) or a pharmaceutical composition containing the same is administered in a generalized manner, for example intravenous, intramuscular, subcutaneous, intra-articular, transdermal, intrarachidian, periosteal, intratumoral, intralesional, perilesionally by infusion (for example, using an infusion device), orally, topically or by inhalation. Preferably, the CD2 binding agent is administered intramuscularly or intravenously. In another embodiment, the CD2 binding agent is administered locally, for example, topically or by injection without needles, into an affected area. Parenteral administration of the binding agent to CD2 (for example, the LFA-3 / lg fusion), the second agent, (or both) or a pharmaceutical composition containing it can be effected using a needle or a needleless syringe by means of methods known in the art.

Examples of syringe systems without needles and modes of administration are described in US 6,132,395, US 6,096,002, US 5,993,412, US 5,893,397, US 5,520,639, US 5,503,627, US 5,399,163, US 5,383,851, US 5,312,577, US 5,312,335, whose contents are incorporated into the present as a reference. Pharmaceutical Compositions Preferably, an effective amount of the CD2-LFA-3 inhibitor (e.g., a soluble CD2-binding LFA-3 polypeptide described herein) is administered. By "effective amount" is meant an amount capable of decreasing the spread or severity of the conditions described herein. In therapeutic modalities, an effective amount of the agent refers to an amount of an agent that is effective, from administration in single or multiple doses to the subject, by inhibiting, reducing or ameliorating the disorder (e.g., improving the PASI evaluation). or PGA assessment for a patient with psoriasis), or by prolonging the survival of the patient with the disorder beyond what is expected in the absence of such treatment. The improvement of psoriasis, for example, is predicted to lead to improved quality of life, which was evaluated, for example, by the SF-36 health questionnaire developed by RAND Health, a division of the RAND Corporation (Santa Monica, CA ). An effective amount does not necessarily indicate a total elimination of the disorder. In prophylactic modalities, an effective amount of an LFA-3 or CD2 binding agent described herein refers to an amount of an agent that is effective, from administration in single or multiple doses to the subject, by preventing or delay the occurrence of the beginning or the recurrence of the disorder. It will be obvious to those skilled in the art that the effective amount of the agent will depend, inter alia, on the disorder treated (e.g., skin T cell mediated disorder Vs T-cell mediated disorder of another organ other than the skin) , the administration program, the unit dose administered, if the agent is administered in combination with other therapeutic agents, the immune status and health of the patient, the therapeutic or prophylactic activity of the particular agent administered and the serum half-life. Depending on the disorder to be treated, the agent can be packaged differently. Preferably, the soluble CD2-binding LFA-3 polypeptide (for example LFA3-TIP) is administered in a dose of between about 0.001 and about 50 mg of the agent per kg of body weight, more preferably, between about 0.01 and about 10. mg of the agent per kg of body weight, mostly between about 0.1 and about 4 mg of the agent per kg of body weight is preferred. In a preferred embodiment, the CD2 binding LFA-3 polypeptide is administered at a unit dosage ranging from 2 to 15 mg when administered by IV route (eg, 7.5 mg IV bolus) and at a dosage ranging from 2 to 30 mg when administered by the IM route (e.g., IM injection of 10 mg or 15 mg). IM and IV administration are preferred. Unit doses are typically administered until an effect is observed. The effect can be measured by a variety of methods, including in vitro T cell activity assays and the removal or improvement of affected skin areas, or improvement in other affected areas of the body that may be relevant to the particular disorder. Preferably, the unit dose is administered at regular intervals during a treatment cycle, such as once a week. More preferably, it is administered at regular intervals, for example, at weekly intervals for a period of administration of several weeks, for example, eleven weeks. More frequent administrations, for example, two or three times a week are also contemplated and can be adopted if the subject's disorder is severe or if urgent intervention is indicated. Less frequent administrations, for example once or twice per month, are also contemplated and can be adopted if the subject responds well to therapy so that maintaining the dosage is appropriate. It will be recognized, however, that higher or lower dosages and other administration schedules may be employed during any one of the particular administration cycles. The agent, for example, the CD2-binding LFA-3 polypeptide (e.g. AMEVIVE) is also preferably administered in a composition that includes a pharmaceutically acceptable carrier. By "pharmaceutically acceptable carrier" is meant a carrier that does not cause an allergic reaction or other undesirable effect in patients in whom it has been administered. Suitable pharmaceutically acceptable carriers include, for example, one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like, as well as combinations thereof. Pharmaceutically acceptable carriers may additionally comprise minimal amounts of auxiliary substances such as emulsifying or wetting agents, preservatives or regulators, which improve the shelf life or effectiveness of the agent. Formulations, for example, pharmaceutical formulations, of CD2 binding agents can be prepared in aqueous or non-aqueous forms, for example lyophilized. Preferred pharmaceutical formulations are suitable for injection. An example of an aqueous formulation encompassed by the present invention includes liquid formulations frozen with phosphate buffered saline (PBS). An example of a lyophilized formulation includes one or more of: citrate, glycine or sucrose. For example, a preferred lyophilized formulation includes 1 to 5% sucrose, preferably 2.5% sucrose, and 0.5% to 2% glycine, preferably 1% glycine, in citric acid-sodium citrate buffer (at least 10 mM) , preferably 25 mM) regulated at pH of at least about 4, preferably 5, more preferably 6 (or even more preferably, 6.8). The second agent can be administered in a single dosage form with the CD2 binding agent (i.e. as part of the same pharmaceutical composition), a multiple dosage form wherein the two components are administered separately and specifically. Alternatively, the CD2 binding agent and the other active agent may be in the form of a single conjugated molecule. The conjugation of the two components can be achieved by means of standard crosslinking techniques well known in the art. A single molecule can also take the form of a recombinant fusion protein. In addition, a pharmaceutical composition useful in the present invention can be used in combination with other therapies such as PUVA, chemotherapy and UV rays. Such combination therapies can advantageously use lower dosages of the therapeutic or prophylactic agents. The CD2 binding agent, or the pharmaceutical composition, can be in a variety of forms. These include, for example, solid, semi-liquid and liquid dosage forms, such as tablets, pills, powders, liquid solutions, dispersions or suspensions, liposomes, suppositories, infusible injectables, and topical preparations. The preferred form depends on the mode of administration and the therapeutic application. Preferred forms are infusible and injectable solutions. The invention includes formulations suitable for use as sunscreens or UV protection applied topically. Preferred embodiments include preparations of AMEVIVE. The active ingredient can be formulated in a liposome. The product can be applied before, during or after exposure to UV rays, or before, during or after the development of redness.

Sequences The following is a summary of the sequences described in US 6,162,432 and mentioned in the course of the application: SEQ ID NO: 1 Transmembrane LFA-3 DNA Sequence SEQ ID NO: 2 Transmembrane LFA-3 amino acid sequence SEQ ID NO: 2 NO: 3 LFA-3 DNA sequence linked to Pl SEQ ID NO: 4 LFA-3 amino acid sequence linked to Pl SEQ ID NO: CD2 DNA sequence SEQ ID NO: 6 CD2 amino acid sequence SEQ ID NO : 7 DNA Sequence of AMEVIVE SEC ID NO: 8 AMEVIVE amino acid sequence EXAMPLES EXAMPLE 1: Treatment in multiple runs of psoriasis with AMEVIVE® (Alefacept) This example examined the efficiency and safety in patients who have received a multiple treatment run of up to nine cycles of therapy with AMEVIVE® for 4.5 years. Treatment The initial treatment cycle in the open label study is referred to as cycle A. Subsequent cycles are marked cycle B, C, D, and so on. Patients received once a week the treatment for 12 weeks (administration period) followed by 12 weeks of observation (rest period) in each cycle. 7.5 mg of AMEVIVE were administered by intravenous (IV) bolus injection. Before the initiation of the treatment in the extension of the study the need for generalized therapy was established based on the evaluations of the severity of the physician's disease, and the CD4 + T cell counts were at or above the lower limit of the normal (LLN 404 cells / mm3). The eligibility of a subsequent cycle was based on the criteria mentioned above and, in addition, patients should have received > 8 doses of AMEVIVE during the 12-week treatment period of the previous cycle and for cycle C and subsequent cycles, the lymphocyte count was required to be outside of > 75% of the account registered in the study selection visit. In any given cycle, if the patients had CD4 + T cell counts < 300 but > 200 cells / mm3, the dose of AMEVIVE was reduced by 50% (3.75 mg). If the CD4 + T cell count was < 200 cells / mm3, the programmed dose was maintained. If the CD4 + T cell count was < 200 cells / mm3 for 4 consecutive visits, AMEVIVE was permanently maintained. The dose of AMEVIVE was retained for 2 weeks when there was evidence of a clinically significant infection. Evaluation The efficiency in the Severity and Psoriasis Area Index (PASI) and the Global Physician Evaluation (PGA) was evaluated. For cycle A, evaluations were made in weeks 1, 3, 5, 7, 9, 11, and 12 during treatment and at 2, 4, 6, 8, and 12 weeks after treatment. For subsequent cycles, evaluations were made in weeks 1 and 7 during treatment and at 2 and 12 weeks after treatment. The proportions of patients who achieved > 50% and > 75% improvement in PASI from baseline (PASI 50 and PASI 75, respectively) and those who achieved PGA by "clear" or "almost clear". Initial analyzes of lymphocytes and total lymphocytes were conducted at each study visit, except at 4 weeks after treatment in cycle A and 4 and 8 weeks after treatment in all subsequent cycles. Patients with viral, bacterial or fungal infections in progress were monitored at all visits. Adverse events were monitored during the course of the study. Results At the time of this analysis, patients had received repeated courses of therapy with AMEVIVE during 4. 5 years. In this study, 175 patients had received > 1 AMEVIVE cycle; 126 received > 2 cycles; 96 received > 3 cycles; and 71 received > 4 cycles Some patients had received up to 9 AMEVIVE cycles as a result of exposure to AMEVIVE in previous two-phase studies. Efficiency The proportions of patients who achieved PASI 50 in 2 or 12 weeks after treatment for the AD cycles are shown in Figure 2. The response speed of the cycles C and s markedly increased compared to cycles A and B. The proportions of patients who achieved PASI 75 were 29 %, 33%, 34%, and 52% in cycles A, B, C, and D, respectively. The corresponding response rates for a "clear" or "almost clear" PGA were 24%, 29%, 33%, and 34%, respectively. For AD cycles, the increased benefit and repeat response of additional cycles of therapy with AMEVIVE are shown in Figure 3. For cycle A responders ie patients who achieved PASI 50 in cycle A) who received additional cycles of AMEVIVE, the proportions of patients who achieved PASI 50 increased with each subsequent cycle. In general, patients continued to respond to the repetition of AMEVIVE treatment without evidence of tachyphylaxis. Of those who achieved PASI 50 in a given cycle, 75% to 90% of patients achieved PASI 50 in subsequent cycles (ie, repeated response). Adverse Events The incidence of adverse events, in general, did not vary considerably through the cycles. The global safety profile of AMEVIVE after multiple cycle administration (a multiple treatment run) is similar to that reported in 3-phase studies. The incidence of serious adverse events was 7% or less in any cycle, and the spectrum of adverse events was similar to previous studies of 2 and 3 phases. Two patients from each of cycles A and B and 1 patient in cycle E interrupted the treatment to accuse of an adverse event. The incidence of malignancy was low: 3% or less in any cycle; most had skin cancer. Duration of the treatment-free response In the 3-phase studies, the remitting action of AMEVIVE was demonstrated, with patients who maintained PASI 50 responses for a mean of 7 months. In this example, some patients had been followed for prolonged periods after successful treatment cycles. Figure 4 shows the maximum length of response time in 4 of said patients. The response to therapy with AMEVIVE was maintained for 18-24 months in some patients. Lymphocyte count Through the multiple treatment cycles with AMEVIVE, the decrease in lymphocyte sequences was consistent. The decrease in the lymphocyte counts observed with each cycle was not cumulative. Mean CD4 + T cell counts remained above LLN for all cycles and did not decrease with exposure to AMEVIVE in multiple runs (Figure 5).

In summary, this study shows that a multiple course of treatment (3 cycles of treatment or more) provides more significant results than a single course of therapy, with no apparent additional risk of side effects. Multiple AMEVIVE cycles were well tolerated by patients, and the incidence of adverse events did not vary significantly through the cycles. Example 2: Treatment in multiple runs of psoriasis with AMEVIVE This example examined the clinical response to a second course of treatment with AMEVIVE in patients with psoriasis who did not achieve a reduction of > 50% in Severity Index and Psoriasis Area (PASI% =) or a reduction > 25% (PASI 25) during a first cycle of treatment with AMEVIVE, as well as the efficiency of multiple therapy cycles. Patients The patients were from > 16 years old and had chronic plaque psoriasis by > 12 months that involved > 10% of the body surface area. The CD4 + T cell counts required to be above the lower limit of the normal (LLN). Treatment with phototherapy, generalized retinoids, generalized corticosteroids, generalized fumarates, immunosuppressants (methotrexate, cyclosporine, azathioprine, and thioguanine), and high potency topical corticosteroids were banned in 4 weeks before treatment with AMEVIVE and during the course of studies. The use of topical corticosteroids of moderate potency, topical retinoids, coal tar, keratolytics, and vitamin D analogues were banned in 2 weeks of treatment with AMEVIVE and in the aftermath of the studies, except on the scalp, groin, and plants. from the feet. To be eligible for enrollment in the extension studies, patients were required to have received >; 8 doses of AMEVIVE and having completed the final follow-up visit of the previous therapy cycle. Patients were excluded from the extension studies if they were enrolled in any other drug therapy research study or without drugs or if they initiated alternative generalized psoriasis treatments, phototherapy, or other disapproved therapies before 8 weeks of the previous cycle Treatment The studies of 3 phases were multiple-center, randomized, double-blind, and placebo-controlled (Krueger et al., J. Am. Acad. Dermatol., 47: 821- 833, 2002; Lebwohl et al., Arch. Dermatol., 139: 719- 727, 2003). In the study of 3 phases of intravenous (IV) treatment with AMEVIVE, patients received two treatment cycles, where each cycle consisted of (i) a 12-week administration period either once a week of AMEVIVE (7.5 mg) or placebo, and (ii) a follow-up of 12 weeks (rest period). In the study of 3 phases of intramuscular (IM) treatment with AMEVIVE, patients received a single treatment cycle consisting of a 12-week administration period of either AMEVIVE (10 mg or 15 mg) or placebo once a week. , followed by 12 weeks of observation (rest period). In multiple-therapy studies, patients who received additional courses of treatment with AMEVIVE (same dosing regimen) were those whose disease had progressed to require generalized therapy or phototherapy, as determined by the researcher, and had CD4 + T cell counts circulating in or on LLN (Gordon et al, J. Drugs Dermatol, 2: 624-628, 2003). Evaluation During the 3-phase studies, PASI and the Physician Global Assessment (PGA) were evaluated baseline, every two weeks during the treatment, and every 2 to 4 weeks during the follow-up. PASI and PGA were also evaluated at several time points during the IV multiple-run study, while only PGA was evaluated during the IM multiple-run study. Analysis The data from the 3-phase study of 2 cycles of IV treatment with AMEVIVE were used to determine the efficiency of a second treatment cycle with AMEVIVE in two groups of patients: (a) those who did not achieve PASI 50 in the first cycle and (b) those who did not achieve PASI 25 in the first cycle. We determined the proportions of patients who did not achieve PASI 50 and PASI 25 during cycle 1 of treatment with AMEVIVE who achieved PASI 50 or reductions of PASI of > 75% (PASI 75) at any time during a second treatment cycle. Abnormal proportions and corresponding 95% confidence intervals (CIs) were calculated to compare the response rates between patients who received a second course of treatment with AMEVIVE and those who received placebo. We also determined the proportions of patients who achieved PASI 50, PASI 75, and PGA of "clear" or "almost clear" at any time during each of the IV treatment cycles, and the proportion of patients who achieved a PGA of "clear" or "almost clear" at any time during each of the IM treatment cycles. Results Patients had a mean age of ~ 45 years, and ~ 70% were males. The average duration of psoriasis was ~ 19 years, and the average surface area involved was ~ 22%. The number of patients who received each IV and IM treatment cycle with AMEVIVE is summarized in Table 1.

Table 1. Number of patients who received multiple cycles of AMEVIVE.

Efficiency of a second cycle of treatment in patients who did not previously achieve PASI 50 or PASI 75 Improvement of PASI was observed during a second cycle of IV treatment with AMEVIVE in 93% of patients who did not achieve PASI 50 during the first cycle and 89% of patients who did not achieve PASI 25 during the first cycle. Treatment with AMEVIVE in the second cycle resulted in significantly higher proportions of patients who achieved PASI 50 and PASI 75 than placebo treatment (Table 2). 19% of patients who did not achieve PASI 50 during the initial course of treatment with AMEVIVE achieved PASI 75 during the second cycle and 53% achieved PASI 50. 14% of patients who did not achieve PASI 25 during the initial treatment cycle with AMEVIVE achieved PASI 75 during the second cycle and 47% achieved PASI 50 (Table 2). Abnormal proportions showed that patients who did not achieve PASI 50 or PASI 25 in cycle 1 were 2 to 3 times more likely to achieve PASI 50 or PASI 75 in cycle 2 compared to patients who did not receive another cycle (Table 2). Table 2. Improvement of PASI at any time during a second cycle of AMEVIVE in patients who did not achieve PASI 50 or PASI 25 during the first cycle.

A = Patients who did not achieve PASI 50 in the first AMEVIVE cycle.

Efficiency of Multiple-Run Therapy Patients who received multiple courses of IV treatment with AMEVIVE showed increasing improvement in PASI during each subsequent treatment cycle. The proportion of patients who achieved PASI 75 increased from 29% during cycle 1 to a maximum of 54% during cycle 5 (Figure 6A). Similarly, the proportion of patients who achieved PASI 50 increased from 56% during cycle 1 to a maximum of 74% during cycle 5 (Figure 6B). PGA was additionally supporting the increase in clinical improvement in psoriasis observed with multiple cycles of treatment with AMEVIVE. 23% of patients had a "clear" or "almost clear" PGA during cycle 1 of IV treatment with AMEVIVE. During cycle 5, 44% of patients achieved this level of response (Figure 7A). For the IM treatment with AMEVIVE, the "clear" / "almost clear" PGA response rates increased from 21% during cycle 1 to a maximum of 41% during cycle 4 (Figure 7B). In summary, increasing clinical improvement was seen after successive courses of treatment with AMEVIVE, indicating its efficiency for the long-term treatment of patients with chronic plaque psoriasis. Multiple-run therapy data showed that the response of patients to additional treatment with AMEVIVE is increasingly beneficial with respect to the response to initial treatment.

Claims (30)

1. NOVELTY OF THE INVENTION Having described the present invention, it is considered as a novelty, and therefore the content of the following is claimed as property: CLAIMS A method of treating a subject having psoriasis, the method is characterized in that it comprises administering a multiple course of treatment of a soluble CD2 binding LFA-3 polypeptide to the subject, because the multiple run comprises multiple treatment cycles, and because each cycle includes a period of administration and a period of rest. 2. The method according to claim 1, characterized in that the soluble CD2 binding LFA-3 polypeptide is an LFA-3 fusion protein. 3. The method according to claim 1, characterized in that the soluble CD2-binding LFA-3 polypeptide is an LFA-3 / immunoglobulin (Ig) fusion protein. 4. The method according to claim 1, characterized in that the LFA-3 polypeptide binding to Soluble CD2 comprises a soluble LFA-3 polypeptide fused to all or part of the joint region of the Ig heavy chain and all or part of a heavy chain constant region. The method according to claim 1, characterized in that the soluble CD2-binding LFA-3 polypeptide comprises a fusion protein consisting of the 92 amino-terminal amino acids of mature LFA-3, the terminal 10 amino acids C of a human IgG1 joint region, a CH2 region of a human IgG1 heavy chain, and at least part of the CH3 region of a human IgG1 heavy chain. 6. The method according to claim 1, characterized in that the soluble CD2 binding LFA-3 polypeptide is AMEVIVE (Figure 1). 7. The method according to claim 1, characterized in that the LFA-3 polypeptide binding to Soluble CD2 is encoded by an insert contained in plasmid pSAB152, deposited with American Type Culture Collection under Accession Number ATCC 68720. 8. The method according to any one of claims 1-7, characterized in that the multiple run comprises the minus four treatment cycles. 9. The method according to any one of claims 1-7, characterized in that the multiple run comprises at least five treatment cycles. 10. The method according to any one of claims 1-7 characterized in that the multiple run comprises at least six treatment cycles. 11. The method according to any one of claims 1-7 characterized in that the multiple run comprises at least seven treatment cycles. 12. The method according to any one of claims 1-7, characterized in that the multiple run comprises at least eight treatment cycles. The method according to any one of claims 1-7 characterized in that the rest period of each successive cycle of the multiple run is longer than the rest period of a previous run in the multiple run. 14. The method according to any one of claims 1-7 characterized in that the rest period of the last cycle of the multiple run is at least 2 years. 15. The method according to any one of claims 1-7, characterized in that the rest period of the last cycle of the multiple run is at least 3 years. 16. The method according to any one of claims 1-7 characterized in that the period of administration of each cycle of the multiple run is at least 8 weeks. 17. The method according to any one of claims 1-7 characterized in that the period of administration of each cycle of the multiple run is at least 10 weeks. 18. The method according to any one of claims 1-7 characterized in that the period of administration of each cycle of the multiple run is at least 12 weeks. 19. The method according to any one of claims 1-7 characterized in that the polypeptide is administered intramuscularly. 20. The method according to any one of claims 1-7 characterized in that the polypeptide is administered intravenously. 21. The method according to any one of claims 1-7 characterized in that the polypeptide is administered in a unit dosage ranging from 2 to 30 mg. 22. The method according to any one of claims 1-7 characterized in that the method further comprises administering to the subject an additional therapeutic or prophylactic agent during the multiple course of treatment. 23. A method of treating a subject in need of treatment for psoriasis, the method is characterized in that it comprises administering a multiple course of AMEVIVE treatment (Figure 1) to the subject, because the multiple course of treatment comprises at least three treatment cycles, each treatment cycle comprising a period of administration administration once per week of AMEVIVE (Figure 1) for 12 weeks, followed by a rest period of at least 12 weeks. The method according to claim 23, characterized in that the treatment multiple run comprises at least four treatment cycles. 25. The method according to claim 23, characterized in that the treatment multiple run comprises at least five treatment cycles. 26. The method according to claim 23, characterized in that the method comprises evaluating the subject for the effects of AMEVIVE (Figure 1) during one or both of the period of administration and the rest period of each cycle in the multiple run. 27. The method according to claim 23, characterized in that the method further comprises administering to the subject an additional therapeutic or prophylactic agent during the multiple course of treatment. 28.A method of treating a subject having psoriasis, the method is characterized in that it comprises (a) selecting a subject on the basis of having had at least two cycles of treatment with a soluble CD2-binding LFA-3 polypeptide and (b) administering a third cycle of treatment of a LFA-3 polypeptide binding to CD2 soluble to the subject. 29. The method according to claim 28, characterized in that the soluble CD2 binding LFA-3 polypeptide is AMEVIVE (Figure 1). 30. An equipment characterized in that it comprises a pharmaceutical composition comprising AMEVIVE and instructions for administering the pharmaceutical composition to a patient who has previously had two courses of treatment with AMEVIVE (Figure 1).